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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2293-2305.)
© 1997 American Heart Association, Inc.

Articles

Evidence for an -Granular Pool of the Cytoskeletal Protein -Actinin in Human Platelets That Redistributes With the Adhesive Glycoprotein Thrombospondin-1 During the Exocytotic Process

Véronique Dubernard; Brigitte B. Arbeille; Monique B. Lemesle; ; Chantal Legrand

From Unité INSERM 353, Hôpital Saint-Louis, Paris (V.D., C.L.), and Centre de Recherches sur le Sang et les Vaisseaux de l'Association Claude Bernard, Hôpital Lariboisière, Paris (V.D.); and Laboratoire de Microscopie Electronique, Faculté de Médecine, Tours, France (B.B.A., M.B.L.).

Correspondence to Dr Véronique Dubernard, Unité INSERM 353, Hôpital Saint-Louis, 1, avenue Claude Vellefaux, 75010-Paris, France.

	Abstract

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Abstract In a previous study, we have demonstrated that the platelet adhesive glycoprotein thrombospondin-1 (TSP-1) interacts specifically with the cytoskeletal protein -actinin in a solid-phase binding assay. Stored in the -granules of platelets, TSP-1 is secreted during cell activation and binds to the plasma membrane promoting the platelet macroaggregate formation. However, the molecular mechanism by which TSP-1 reaches and binds to the platelet surface is to date unelucidated. -Actinin is an actin-binding and actinin–cross-linking protein that is present in most cells and may act as a link between the bundles of F-actin and the plasma membrane. In this study, we have investigated a possible interaction of -actinin with TSP-1 in platelets by examining their respective subcellular location during the platelet activation process. By indirect immunofluorescence, -actinin was found to display a granular staining in resting platelets similar to that of TSP-1. Performing postembedding immunogold labeling for electron microscopy, we detected the presence of -actinin throughout the cytoplasm, but the strongest gold staining was found in organelles identified as -granules on the basis of their ultrastructure and TSP-1 content. With the use of double immunogold labeling on platelets at different stages of activation by thrombin, both -actinin and TSP-1 were seen redistributing from the -granules to the platelet surface via the open canalicular system (OCS). At the same time, the cytoplasmic -actinin concentrated toward the plasma membrane, but no colocalization with the F-actin bundles was evidenced. Finally, preembedding immunogold labeling and immunoprecipitation of ¹²⁵I-surface–labeled, thrombin-activated platelets further demonstrated that -actinin was expressed on the plasma membrane in the absence of any detectable expression of actin and that it could form molecular complexes with TSP-1 on activated platelets. These results suggest that -actinin found to be present on the platelet surface together with TSP-1 originates in the -granules by fusion of the -granules with the plasma membrane during platelet exocytosis.

Key Words: thrombospondin-1 • -actinin • -granule • platelet exocytosis • molecular complexes

	Introduction

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Blood platelets play an essential role in the first phase of the hemostatic process through their ability to adhere to the damaged vessel wall and to be activated and aggregate in response to various agonists. An impaired platelet activation process and/or impaired adhesive functions may result in hemorrhagic or thrombotic diseases.^{1 2 3} On a molecular basis, the interaction of the adhesive glycoprotein fibrinogen with its platelet receptor, the integrin _IIbß₃, plays a pivotal role in the initiation of platelet aggregation by cross-linking adjacent platelets,⁴ yet additional adhesive glycoproteins such as von Willebrand factor (vWF) and TSP are important for platelet cohesion and macroaggregate formation.^{3 5} Platelet TSP, or TSP-1, is a 420-kD homotrimer and the prototypic member of a family of related extracellular matrix molecules that may modulate cell adhesion, migration, and proliferation in several physiopathological processes such as platelet aggregation, angiogenesis, atherosclerosis, and tumor metastasis.^{6 7 8 9} Blood platelets contain a high amount of TSP-1,¹⁰ which is rapidly secreted from the -granules upon platelet activation together with other adhesive glycoproteins (eg, fibrinogen, fibronectin, and vWF).^{11 12 13 14} TSP-1 binds to the surface of activated platelets and mediates interaction of platelets with each other^{5 15 16 17 18} and with other circulating blood cells such as monocytes.¹⁹ TSP-1 is also incorporated into the fibrin clot network and may regulate its degradation by interacting with components of the fibrinolytic system.^{20 21} Despite so many studies emphasizing the role of TSP-1 in hemostasis, little is known about the molecular mechanism by which TSP-1 is secreted from the -granules to the platelet surface and interacts with the surface of activated platelets. Membrane-bound fibrinogen and CD36, also named GP IV, have been identified as potential TSP-1–binding molecules on the surface of activated platelets,^{5 16 17 22 23} yet this is to date controversial.^{24 25 26} In a quest to identify platelet TSP-1-binding molecules with ligand blot and solid-phase binding assays, we have shown in a previous study a specific and high affinity interaction of TSP-1 with the cytoskeletal protein -actinin.²⁷ The objective of the present work was to investigate whether such a molecular interaction could occur in a cellular context. For this, we used a morphologic approach to examine the distribution of -actinin and TSP-1 in resting platelets and during the exocytotic process of platelet activation by thrombin.

-Actinin is an F-actin–binding and F-actin–cross-linking protein found in most cells along actin stress fibers and at sites where the actin microfilaments (F-actin) are anchored such as the Z bands in striated muscle cells, focal adhesions, and intercellular adherens junctions in other cells.^{28 29 30} The native protein is a homodimer with subunit molecular mass of 100 kD arranged in an antiparallel fashion,²⁹ but several distinct isoforms of -actinin have been characterized on the basis of structural and immunologic differences.^{29 31 32} As yet, however, the only clear functional difference between these isoforms is that nonmuscle -actinins, unlike their muscle counterparts, bind to actin filaments in a calcium-sensitive manner.^{29 33} On the basis of a number of studies showing -actinin to associate with plasma membrane lipids,^{34 35 36 37} cytoskeletal proteins,²⁸ and cytoplasmic domains of adhesion receptors,^{38 39 40 41} -actinin is postulated to be a link between the F-actin cytoskeleton and the plasma membrane either directly or via a series of protein interactions.^{28 30} In platelets, -actinin has been identified as a component of the actin cytoskeleton^{42 43 44} and has been localized in the cytoplasm from which it redistributes during platelet activation toward the plasma membrane, filling up broad pseudopods.^{45 46 47}

In this study, we demonstrate that in addition to its localization in the cytoplasm, -actinin is also found in the -granules of human platelets and that during platelet activation this -granular -actinin redistributes to the cell surface in a similar manner to TSP-1. Evidence is also provided for formation of molecular complexes between -actinin and TSP-1 on the plasma membrane of thrombin-activated platelets.

	Methods

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Preparation and Characterization of Antibodies
The mouse anti-human platelet -actinin ascitic fluid was purchased from Chemicon International and the rabbit anti-human platelet -actinin serum was generously provided by Dr Françoise Landon and Dr Yannick Gache (College de France, Paris). The immunoglobulins were purified by affinity chromatography on Protein G Sepharose 4 Fast Flow (Pharmacia Biotech) according to standard procedures and were characterized by Western immunoblotting with purified -actinin isolated from human platelets as described elsewhere.²⁷ The mouse anti–TSP-1 ascitic fluid MAII and the rabbit anti–TSP-1 serum R1 were a kind gift of Prof Jack Lawler (Harvard Medical School, Boston, Mass). The preparation and characterization of the purified IgGs have been reported.^{17 48} Purified IgGs from a rabbit antiserum to -smooth muscle actin were kindly provided by Prof Giulio Gabbiani (Faculté de Médecine, Genève, Switzerland), and the rabbit antisera to human platelet GP IIb and GP IIIa were a gift from Dr Dominique Pidard (Institut Pasteur, Paris, France). IgGs purified from a rabbit preimmune serum were used as control polyclonal IgGs, and MOPC21, a mouse myeloma IgG1 (Sigma Chemical Co), was used as irrelevant monoclonal IgG1.

Platelet Preparation and Cytoskeleton Isolation
Platelets were isolated from freshly drawn human blood collected from aspirin-free adult donors into acid-citrate-dextrose and washed by repeated centrifugations in a modified Tyrode's buffer, pH 6.5, supplemented with 3.5 mg/mL BSA (Fraction V, Sigma), 25 µg/mL apyrase (Grade I, Sigma), and 100 nmol/L prostaglandin E₁ (PGE₁) (Sigma), as described elsewhere.²⁴ The final pellet was resuspended at a concentration of 3x10⁸ platelets/mL in a 5 mmol/L HEPES-buffered Tyrode's solution (137 mmol/L NaCl, 3 mmol/L KCl, 12 mmol/L NaHCO₃, 0.36 mmol/L NaH₂PO₄, 5.5 mmol/L glucose, 2 mmol/L CaCl₂, and 1 mmol/L MgCl₂), pH 7.4, containing 3.5 mg/mL BSA. Platelets were activated at 37°C with 0.1 U/mL thrombin (3,000 NIH U/mg protein, Sigma), and platelet lysis was quantified by the release of lactate dehydrogenase. Under all experimental conditions, minimal lactate dehydrogenase release was measured that was not increased upon thrombin activation (1.7±0.2%, mean±SEM, n=4) when compared with that of resting platelets (2.0±0.3%).

The actin cytoskeleton from resting and thrombin-activated platelets was prepared as follows: washed platelets were either supplemented with inhibitors of activation (ie, 25 µg/mL apyrase and 100 nmol/L PGE₁) or activated without stirring for 5 minutes at 37°C with 0.1 U/mL thrombin. Activation was stopped by the addition of a 20-fold (U/U) excess of hirudin (2200 U/mg protein, Sigma). Platelets were sedimented by centrifugation at 1300g for 15 minutes and solubilized at 2x10⁹ cells/mL by agitation for 30 minutes at 4°C in 15 mmol/L Tris, 150 mmol/L NaCl, pH 7.4, containing 1% (vol/vol) Triton X-100 (Sigma), 1 mmol/L EDTA, 0.2 mmol/L leupeptin (Sigma), and 1 mmol/L benzamidine (Sigma). The Triton X-100 insoluble cytoskeletal fraction was recovered by centrifugation of the Triton X-100 lysate for 5 minutes at 16 000g. The resulting pellet was resuspended in a volume equivalent to that of the Triton X-100 platelet lysate and solubilized by heating at 100°C for 5 minutes in the presence of 2% (wt/vol) SDS and 5% (vol/vol) ß-mercaptoethanol, as previously described.²⁴

Surface Iodination of Platelets and Immunoprecipitation Experiments
Washed platelets were resuspended at 5x10⁸ cells/mL in Tyrode's buffer, pH 7.4, in which BSA was omitted. Unactivated platelets supplemented with inhibitors of platelet activation (PGE₁ and apyrase) and thrombin-activated platelets prepared as described above were surface-labeled by lactoperoxidase-catalyzed iodination with the use of the method of Phillips and Agin (1977).⁴⁹ Briefly, 1 mCi of carrier-free Na ¹²⁵I (CIS Bio International) was added to 4 mL of thrombin-activated platelets (2x10⁹ cells) followed by 10 µL of 0.25 mmol/L lactoperoxidase (Sigma) and 5x10 µL of freshly prepared 1 mmol/L H₂O₂, added at 10-second intervals. The radiolabeled platelets were diluted 5-fold and washed three times in Tyrode's buffer, pH 6.5, supplemented with 3.5 mg/mL BSA and 25 µg/mL apyrase. Platelets were lysed at 2x10⁹ cells/mL by agitation for 30 minutes at 4°C in Tyrode's buffer, pH 7.4, containing 1% (vol/vol) Triton X-100, 0.2 mmol/L leupeptin, and 1 mmol/L benzamidine (immunoprecipitation buffer). The Triton X-100-insoluble materiel was removed by centrifugation of the platelet lysate for 15 minutes at 16 000g, and the supernatant was frozen at -80°C. Immunoprecipitation experiments were performed with thawed samples cleared by centrifugation for 15 minutes at 16 000g. Proteins (100 µg) from the platelet lysate were incubated for 60 minutes at 4°C with each of the following antibodies: 10 µg of the monoclonal antibody to -actinin, 10 µL of the mouse anti–TSP-1 ascitic fluid (MAII), 50 µg of the polyclonal antibody to -actinin, TSP-1 (R1), or actin. Samples were then incubated for 60 minutes at 4°C with Protein G Sepharose 4 Fast Flow gel, equilibrated in the immunoprecipitation buffer and saturated with 2% BSA, then centrifuged for 1 minute at 12 000g. The Sepharose beads were washed three times in the immunoprecipitation buffer by centrifugation for 10 seconds at 12 000g, and the immune complexes were eluted from beads by heating at 100°C for 5 minutes in the presence of 2% (wt/vol) SDS and 5% (vol/vol) ß-mercaptoethanol. After centrifugation for 1 minute at 12 000g, the whole supernatants were analyzed by electrophoresis on a 7% to 12% exponential gradient polyacrylamide gel followed by autoradiography using Kodak X-Omat films (Kodak-Pathé).

SDS-PAGE and Western Immunoblotting
Solubilized platelet proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue, as described elsewhere.²⁴ All reagents for SDS-PAGE were purchased from Bio-Rad.

For Western immunoblotting, SDS-PAGE separated platelet proteins were electrotransferred onto nitrocellulose sheets and probed with 10 µg/mL of the polyclonal or monoclonal antibody to -actinin, or the polyclonal antibody to actin, or with a 1/1000 dilution of the rabbit antisera to GP IIb and GP IIIa, followed by incubation with ¹²⁵I-Protein A (Amersham) and autoradiography, as described.²⁴ Nitrocellulose strips probed with the monoclonal antibody were incubated with 10 µg/mL of a rabbit anti-mouse IgG (RAM/7S, Nordic Immunology) before being incubated with ¹²⁵I-Protein A. The relative amount of -actinin in the Triton X-100-soluble fraction and the Triton X-100-insoluble cytoskeletal fraction was quantified by densitometric scanning of autoradiographs with the use of a computer-based image analysis system (Biocom).

Indirect Immunofluorescence
Platelet samples at a concentration of 3x10⁸ cells/mL in Tyrode's buffer, pH 7.4, were supplemented with 25 µg/mL apyrase and 100 nmol/L PGE₁ and fixed by the addition of 2% (wt/vol) paraformaldehyde in 100 mmol/L sodium phosphate buffer, pH 7.4, for 10 minutes at 20°C. Platelets were sedimented by centrifugation for 10 minutes at 1300g, resuspended in Tyrode's buffer to a concentration of 10⁸ cells/mL, and allowed to settle on poly-l-lysine (Sigma)–coated glass coverslips. Attached cells were permeabilized with 0.1% (vol/vol) Triton X-100 for 3 minutes, washed once in 10 mmol/L PBS, pH 7.4, and incubated in PBS containing 1% (wt/vol) BSA (PBS-1% BSA) with each of various monoclonal and polyclonal antibodies used at 20 µg/mL and 50 µg/mL, respectively. After 60 minutes at 20°C, the slides were rinsed three times and incubated in PBS-1% BSA with a 1/50 dilution of rhodamine-conjugated rabbit anti-mouse IgG or rhodamine-conjugated swine anti-rabbit IgG (DAKO A/S) for additional 60 minutes, then washed again. Immunostained platelets were viewed using a fluorescence microscope equipped with a 100x Plan-Neofluor objective (Carl Zeiss). Control experiments were carried out by omitting primary antibody.

Immunoelectron Microscopy
Postembedding Immunogold Labeling
Washed platelets, either unstimulated or stimulated for 1 minute and 5 minutes at 37°C with 0.1 U/mL thrombin, were fixed by the addition of 2% (wt/vol) paraformaldehyde and 0.1% (vol/vol) glutaraldehyde in 100 mmol/L sodium phosphate buffer, pH 7.4, for 10 minutes at 20°C. Fixed platelets were washed twice in PBS-0.35% BSA by centrifugation for 5 minutes at 1300g, dehydrated in graded ethanol, and embedded in LR White medium resin (Taab Lab Equipment). Polymerization was performed for 48 hours at -20°C. Ultrathin sections (70 nm) were cut with a Reichert OM-U3 ultramicrotome (Reichert Scientific Instruments) and mounted on collodion-coated 200- or 300-mesh, thin-bar gold grids (Biocell Research Lab). Single or double labeling was carried out by an indirect immunogold procedure essentially as previously described.⁵⁰

For single labeling, platelet sections were incubated with 40 µg/mL of the polyclonal antibody to -actinin, TSP-1, or actin, diluted in PBS-1% BSA, for 60 minutes at 20°C. Grids were washed several times with PBS-0.1% BSA, then incubated with a 1/30 dilution of goat anti-rabbit IgG conjugated to 15 nm gold particles (Janssen Life Sciences Products) for 40 minutes at 20°C, and washed extensively with PBS-0.1% and distilled water.

For double labeling, platelet sections were labeled with the same anti–TSP-1 or anti-actin antibody revealed with a 5 nm gold-conjugated goat anti-rabbit IgG (Janssen), then labeled with 40 µg/mL of the monoclonal antibody to -actinin revealed with a 15-nm gold-conjugated goat anti-mouse IgG (Janssen).

Preembedding Immunogold Labeling
For preembedding labeling, platelets either unstimulated or stimulated for 5 minutes with thrombin as described above, were prefixed with 0.5% (vol/vol) glutaraldehyde for 10 minutes at 20°C, washed twice in PBS-0.1% BSA, then incubated at 3x10⁸ platelets/mL in PBS-1% BSA with 50 µg/mL of the monoclonal or polyclonal antibody to -actinin, for 60 minutes at 20°C. Platelets were washed twice and incubated at 6x10⁸ cells/mL with a 1/15 dilution of 10 nm gold-conjugated goat anti-mouse or 30 nm gold-conjugated goat anti-rabbit IgG for 60 minutes at 20°C. Incubation with gold conjugates was continued overnight at 4°C, after which platelet suspensions were washed twice and fixed at 3x10⁸ cells/mL in PBS-0.1% BSA with 1.5% (vol/vol) glutaraldehyde for 10 minutes at 20°C. After extensive washing, platelets were postfixed in 1% (wt/vol) osmium tetroxide for 60 minutes at 20°C, dehydrated in graded ethanol, and embedded in Epon 812 resin (Fluka). Polymerization was performed for 48 hours at 60°C.

All specimens were counterstained with aqueous uranyl acetate and lead citrate and examined in a TEM Jeol JEM 1010 electron microscope.

	Results

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Distribution of -Actinin in Platelets by Indirect Immunofluorescence
Immunofluorescence studies were carried out to examine the subcellular localization pattern of -actinin in resting platelets in comparison to that of TSP-1 and actin. Both monoclonal and polyclonal antibodies to human platelet -actinin were used in this study, and the specificity of these antibodies were analyzed by Western immunoblotting (Fig 1). Both antibodies recognized purified platelet -actinin (Fig 1B, lanes 1 and 3), and in the platelet lysate only one major band corresponding to -actinin was observed (Fig 1B, lanes 2 and 4).

View larger version (59K): [in this window] [in a new window]   Figure 1. Characterization of the antibodies to human platelet -actinin by Western immunoblot analysis. Purified platelet -actinin (5 µg) and proteins (30 µg) from a platelet lysate were electrophoresed on a 7% polyacrylamide slab gel under reduced conditions. A, Proteins were stained by Coomassie blue: purified platelet -actinin (lane 1), platelet lysate (lane 2). B, Proteins were electrotransferred onto nitrocellulose sheets and incubated with 10 µg/mL of the polyclonal (lanes 1 and 2) or the monoclonal (lanes 3 and 4) antibody to platelet -actinin. Both antibodies strongly reacted with purified platelet -actinin (lanes 1 and 3) and recognized a single major band of a molecular mass corresponding to that of -actinin in the platelet lysate (lanes 2 and 4).

With the use of the monoclonal antibody to -actinin, resting platelets were found to exhibit diffuse as well as granular staining for -actinin (Fig 2a), which contrasted with the homogeneous cytoplasmic staining observed for actin on the same platelet preparation (Fig 2c). The granular distribution of -actinin looked very similar to that of TSP-1, which is known to be present in the -granules of platelets (Fig 2b). A possible activation-dependent redistribution of these proteins during the experimental course was minimized by the addition of PGE₁ and apyrase, inhibitors of platelet activation. Identical results were obtained for the distribution of -actinin in platelets with the use of the polyclonal antibody to -actinin (data not shown). In control experiments performed by omitting the primary antibody, platelets displayed no labeling (Fig 2d).

View larger version (91K): [in this window] [in a new window]   Figure 2. Comparison of the distribution pattern of -actinin, TSP-1, and actin in resting platelets by indirect immunofluorescence. Washed platelets were fixed, permeabilized, and incubated with the monoclonal antibody to -actinin (a), TSP-1 (b), or the polyclonal antibody to actin (c). In addition to a diffuse staining, the granular immunostaining pattern observed for -actinin in resting platelets was suggestive of an -granular localization when compared with that of TSP-1 (a compared with b). By comparison, the immunostaining of actin in resting platelets appeared homogeneous (c). No immunofluorescence staining was seen in control experiments performed without primary antibody (d). Bar, 5 µm. These results are representative of six reproducible experiments performed on different platelet preparations with either the monoclonal or the polyclonal antibody to platelet -actinin.

Ultrastructural Localization of -Actinin in Platelets by Immunoelectron Microscopy
To get further insight into the subcellular distribution of -actinin in platelets, we analyzed its ultrastructural localization by electron microscopy by using postembedding immunogold labeling.

Resting Platelets
A series of sections of resting platelets were incubated separately with the polyclonal antibody to -actinin, TSP-1, or actin (Fig 3). We observed that -actinin (Fig 3a) was localized throughout the cytoplasm, but the strongest staining was found associated with organelles identified as -granules on the basis of their size, number, and labeling for TSP-1 antigen (Fig 3b). A scattered distribution of -actinin over the matrix of -granules was observed as for TSP-1 (Fig 3, c compared with b). By comparison, actin was never seen located in association with -granules but over the entire cytosol (Fig 3d). In control experiments performed by omitting the primary antibody, no gold labeling was observed (Fig 3e). To confirm the colocalization of -actinin and TSP-1 within the -granules of resting platelets, we performed a double immunogold labeling using the monoclonal antibody to platelet -actinin and the polyclonal antibody to TSP-1 (Fig 4). A weaker labeling of -actinin was obtained with the monoclonal antibody showing a more eccentric location of the protein in the -granules (arrowheads on Fig 4). However, statistical analysis of gold labeling on serial sections clearly indicated that -actinin was located inside the -granules.

View larger version (141K): [in this window] [in a new window]   Figure 3. Ultrastructural localization of -actinin (a and c) compared with that of TSP-1 (b) and actin (d) in resting platelets. Postembedding single immunogold labeling on ultrathin platelet sections was performed with the use of polyclonal antibodies. Electron micrographs a and c show a strong labeling of -actinin in most -granules (-G, arrowheads) in addition to a weaker labeling in the cytoplasm (cy). By comparison, TSP-1 (b) was exclusively located in the -granules, whereas actin was located over the entire cytosol (d). No labeling was observed in control experiments carried out by omitting the primary antibody (e). Bar, 200 nm.

View larger version (119K): [in this window] [in a new window]   Figure 4. Ultrastructural localization of -actinin and TSP-1 in the -granules of resting platelets. Postembedding double immunogold labeling on platelet sections was performed with the use of the monoclonal antibody to platelet -actinin, which was revealed with a 15-nm, gold-conjugated goat anti-mouse antibody and then with the polyclonal antibody to TSP-1, which was revealed with a 5-nm, gold-conjugated goat anti-rabbit antibody. Although it was weaker with the monoclonal antibody, labeling of -actinin was observed in most -granules (arrowheads), where it contrasted to the dense overall labeling of TSP-1 observed with the polyclonal antibody (arrows). Bar, 50 nm.

Thrombin-Activated Platelets
We next examined the redistribution of -actinin during platelet exocytosis induced by cell activation with thrombin. Platelets were activated under nonstirring conditions for 1 minute and 5 minutes at 37°C with 0.1 U/mL thrombin, and activation was stopped by addition of the fixative.

Single immunogold labeling was performed on sections of platelets activated for 1 minute with thrombin with the use of the polyclonal antibody to -actinin, TSP-1, or actin. As illustrated in Fig 5, platelets activated for 1 minute with thrombin had undergone shape change and exhibited nascent broad based pseudopods. -Granules were centralized, and some were seen fused with each other or with the OCS originating from plasma membrane invaginations, in which they discharged their content, thus creating secretion areas. -Actinin was detected in the peripheral area of the cytoplasm (Fig 5b, arrowheads) and in the -granules, where it was found to redistribute to the secretion areas and appeared to concentrate along the membrane of the OCS (Fig 5, a and b). Of particular interest was the immunogold staining of TSP-1 showing a similar association with the membrane of the OCS, thereby following the route of -actinin (Fig 5c, arrowheads). In contrast to the -actinin and TSP-1 localization, actin was seen concentrated in the cytoplasmic area surrounding the centralized secretory granules and toward the plasma membrane, filling up pseudopods (Fig 5d, arrowheads). No labeling was observed when omitting the primary antibody (Fig 5e).

View larger version (125K): [in this window] [in a new window]   Figure 5. Ultrastructural localization of -actinin (a and b) compared with that of TSP-1 (c) and actin (d) in thrombin-activated platelets at an early stage of activation. Platelets were activated for 1 minute with 0.1 U/mL thrombin, and postembedding single immunogold labeling was performed with the polyclonal antibodies. At this stage of platelet activation, most of the -granules were fused with each other (-G) and with the OCS, where they discharged their content, creating secretion areas (S). -Actinin in the -granules redistributed to these secretion areas and associated with the membrane of the OCS (a and b) similarly to TSP-1 (c), whereas actin redistributed toward the plasma membrane filling up pseudopods (d). Cytoplasmic -actinin (cy) was detected near the plasma membrane (b, arrowheads). Control experiments performed without the primary antibody displayed no labeling (e). Bar, 200 nm.

Double immunogold labeling experiments performed at 1 minute and 5 minutes activation using the monoclonal antibody to -actinin and the polyclonal antibody to TSP-1 confirmed the common fate of these two proteins (Fig 6, a and b) that were seen to colocalize in secretion areas (S) and at the cell surface at the area of OCS discharge (Fig 6, b and c; arrowheads). Cytoplasmic -actinin was particularly visible at the cell periphery (Fig 6, a, b, and d; arrows). However, double immunogold labeling performed for -actinin and actin clearly detected the presence of bundles of F-actin (Fig 6, e and f; arrowheads), but no close localization of F-actin with the cytoplasmic -actinin (Fig 6, e and f; arrows) was evidenced at the plasma membrane.

View larger version (140K): [in this window] [in a new window]   Figure 6. Ultrastructural localization of -actinin and TSP-1 (a through d) or -actinin and actin (e, f) in thrombin-activated platelets by double immunogold labeling. Platelets were activated for 1 minute (a) and 5 minutes (b through f) with 0.1 U/mL thrombin, and postembedding double immunogold labeling was performed on platelet sections with either the monoclonal antibody to -actinin and the polyclonal antibody to TSP-1 (a through d) or the monoclonal antibody to -actinin and the polyclonal antibody to actin (e and f). The secretion route of -granular -actinin (15-nm gold particles) and TSP-1 (5-nm gold particles) appeared similar during platelet exocytosis (a and b). At 5 minutes of activation when no intact -granules could be seen, the two proteins colocalized along the secretion areas (S) and at the cell surface (b and c, arrowheads). Simultaneously, the cytoplasmic -actinin (cy) redistributed to the plasma membrane (a, b, and d; arrows). However, as illustrated on the micrographs e and f, no colocalization of the cytoplasmic -actinin (arrows) with F-actin (arrowheads) was visible at the plasma membrane. Bar, 200 nm (a and b) and 100 nm (c through f).

Three and two reproducible experiments were performed for single and double immunogold labeling, respectively, on different platelet preparations.

Analysis of -Actinin in the Actin Cytoskeleton by Western Immunoblotting
Using a biochemical approach, we evaluated the association of the cytoplasmic -actinin with the actin cytoskeleton of resting and thrombin-activated platelets. Platelets, either unstimulated or activated with 0.1 U/mL thrombin under nonaggregating conditions, were solubilized by Triton X-100 in the presence of protease inhibitors and absence of calcium, and the cytoskeleton was recovered by centrifugation of the lysate at low g forces, essentially as described by Fox et al (1988).⁵¹ When examined by SDS-PAGE and Coomassie blue staining, the cytoskeleton of resting platelets appeared to be composed mainly of filamin, -actinin, and actin (Fig 7A, lane 3). After platelet activation by thrombin, increased incorporation of filamin as well as incorporation of myosin and other new proteins in the cytoskeleton were observed, whereas no apparent increase of -actinin was noted (Fig 7A, lane 6). By densitometric scanning of autoradiographs, we estimated that 70% of -actinin was associated with the actin cytoskeleton of either resting or thrombin-activated platelets (Fig 7B, lanes 3 and 6). These results were in accord with the morphologic observations showing that upon platelet activation, the cytoplasmic -actinin redistributing to the plasma membrane was not seen in close localization with F-actin (compare with Fig 6, e and f).

View larger version (67K): [in this window] [in a new window]   Figure 7. Incorporation of -actinin into the actin cytoskeleton. The actin-rich Triton X-100-insoluble cytoskeleton was prepared from resting and thrombin-activated platelets as described in "Methods." Protein samples from the Triton X-100 platelet lysate (lanes 1 and 4), Triton X-100-soluble fraction (lanes 2 and 5), and Triton X-100 cytoskeletal fraction (lanes 3 and 6) from resting (lanes 1 through 3) and thrombin-activated platelets (lanes 4 through 6) were electrophoresed on a 7% to 12% exponential gradient polyacrylamide slab gel and either stained with Coomassie blue (A) or analyzed by Western immunoblotting for the presence of -actinin with the use of the polyclonal antibody (B). Fifteen microliters of the Triton X-100 platelet lysate, 15 µL of the Triton X-100–soluble fraction, and 45 µL of the Triton X-100-insoluble fraction were loaded in A, whereas the same volumes (15 µL) for all the Triton X-100 fractions were loaded in B. As demonstrated in B, the amount of -actinin incorporated into the actin cytoskeleton of resting platelets (lane 3) was not increased after platelet activation by thrombin (lane 6).

Demonstration of the Surface Exposure of -Actinin and Formation of Complexes With TSP-1 on Thrombin-Activated Platelets
Preembedding Immunogold Labeling
To demonstrate the surface expression of -actinin during platelet activation and exocytosis, we carried out a preembedding immunogold labeling procedure. Unstirred samples of resting and thrombin-activated platelets were fixed with glutaraldehyde and incubated with the monoclonal or polyclonal antibody to -actinin followed by the immunogold conjugate, before being processed for electron microscopy. As illustrated in Fig 8 (micrographs b and c), both antibodies clearly detected -actinin on the surface of thrombin-activated platelets, particularly on pseudopods. By comparison, resting platelets showed only occasional binding of gold particles (Fig 8a) that looked very similar to that obtained by omitting the primary antibody or by incubating resting or thrombin-activated platelets with an irrelevant mouse monoclonal antibody (data not shown).

View larger version (96K): [in this window] [in a new window]   Figure 8. Membrane exposure of -actinin on thrombin-activated platelets as demonstrated by preembedding immunogold labeling. Platelets, either unstimulated (a) or stimulated for 5 minutes with 0.1 U/mL thrombin (b and c), were fixed and incubated with the polyclonal (a and b, 30-nm gold particles) or the monoclonal (c, 10-nm gold particles) antibody to platelet -actinin before the embedding procedure, as described in "Methods." Only faint gold labeling was found on resting platelets (a), whereas membrane exposure of -actinin was clearly detected on thrombin-activated platelets, particularly on pseudopods (b and c, arrowheads). Bar, 200 nm.

Radioimmunoprecipitation
As a complementary approach to demonstrate the surface exposure of -actinin on activated platelets, we performed radioimmunoprecipitation experiments by using Triton X-100-solubilized samples prepared from ¹²⁵I-surface-labeled platelets. With the use of the monoclonal antibody to -actinin, the immunoprecipitate obtained from ¹²⁵I-surface–labeled, thrombin-stimulated platelets was found to contain a substantial amount of the ¹²⁵I-radiolabeled protein (Fig 9A, lane 4) that was otherwise barely detectable in the immunoprecipitate obtained from ¹²⁵I-surface-labeled resting platelets (Fig 9A, lane 3). This result demonstrated the surface expression of -actinin during platelet activation with thrombin that correlated with the surface expression of TSP-1 as demonstrated using the monoclonal antibody MAII (Fig 9A, lane 5), whereas no radiolabeled TSP-1 was detected on resting platelets (data not shown). In contrast, thrombin-activated platelets displayed no radiolabeled actin in the immunoprecipitate obtained with the use of the polyclonal antibody to actin, indicating that no membrane exposure of the cytosolic protein occurred during platelet activation and radiolabeling (Fig 9A, lane 9). The presence of -actinin in the immunoprecipitate obtained with the monoclonal antibody to -actinin was verified by Western immunoblotting of the immunoprecipitate similarly obtained from unlabeled thrombin-activated platelets (Fig 9B, lane 2). This immunoprecipitate was also probed with a mixture of rabbit antisera to human GP IIb and GP IIIa because GP IIIa is an abundant and heavily ¹²⁵I-labeled platelet glycoprotein with a molecular mass close to that of -actinin. No GP IIIa was detected in the immunoprecipitate (Fig 9B, lane 4), and no confusion between -actinin and GP IIIa was possible because these proteins exhibited distinct electrophoretic migrations under our experimental conditions (Fig 9B, lanes 1 and 3).

View larger version (39K): [in this window] [in a new window]   Figure 9. Membrane exposure of -actinin on thrombin-activated platelets and formation of complexes with TSP-1 as demonstrated by radioimmunoprecipitation. A, Platelets, either unactivated (lane 1) or activated for 5 minutes with 0.1 U/mL thrombin (lane 2), were surface-labeled with 125iodine, lysed by Triton X-100, and the soluble fractions from unactivated (lane 3) and activated (lane 4) platelets were incubated with the monoclonal antibody to -actinin and immunoprecipitated as described in "Methods." Surface-labeled, thrombin-activated platelets were also incubated with the monoclonal antibody to TSP-1 (lane 5) or an irrelevant monoclonal antibody (lane 6) or with the polyclonal antibody to -actinin (lane 7), TSP-1 (lane 8), actin (lane 9), or a control polyclonal antibody (lane 10). Immune complexes were analyzed by electrophoresis on a 7% to 12% exponential gradient polyacrylamide gel and autoradiography. 125I-labeled -actinin (lane 4) and 125I-labeled-TSP-1 (lane 5) were immunoprecipitated by their respective monoclonal antibody indicating that -actinin and TSP-1 were simultaneously expressed on thrombin-activated platelets. In contrast, no surface expression of actin was evidenced (lane 9). With the use of polyclonal antibodies, 125I-labeled--actinin and 125I-labeled-TSP-1 were coimmunoprecipitated (lanes 7 and 8), demonstrating that molecular complexes between the two proteins were formed on the surface of activated platelets. B, Western immunoblot analysis of the immune complexes obtained with the monoclonal antibody to -actinin with unlabeled thrombin-activated platelets demonstrated the presence of -actinin (lane 2) and the absence of GP IIIa (lane 4) in the immune complexes. Whole proteins from a control platelet lysate (lanes 1 and 3, 20 µg) and the immune complexes (lanes 2 and 4) were probed either with the monoclonal antibody to -actinin followed by a rabbit anti-mouse IgG (RAM) and 125I-Protein A (lanes 1 and 2) or with a mixture of rabbit antisera to GP IIb and GP IIIa and 125I-Protein A (lanes 3 and 4), respectively. On lane 2, bands at 50 and 25 kD in the immune complexes correspond to the reactivity of the heavy and light chains of the monoclonal anti--actinin IgG with the RAM. On lane 5 is illustrated a platelet lysate blotted with the polyclonal antibody to actin showing the electrophoretic position of the protein. Radioimmunoprecipitation experiments are representative of two reproducible experiments.

To investigate the potential presence of molecular complexes of -actinin and TSP-1 on the platelet surface, radioimmunoprecipitation experiments were preferably performed by incubating ¹²⁵I-surface–labeled, thrombin-activated platelets with the polyclonal antibodies to -actinin and TSP-1. Actually, when tested in solid-phase binding assays, the monoclonal antibody to -actinin displayed an inhibitory effect on the interaction of fluid-phase TSP-1 with coated -actinin (V. Dubernard and C. Legrand, unpublished data, 1995). Thus this antibody may not be suitable to detect formation of complexes between the two proteins because its epitope on -actinin would be blocked by TSP-1 within the complex. Then, with the use of the polyclonal antibodies, radioimmunoprecipitation experiments clearly showed the presence of two radiolabeled bands in the position of -actinin and TSP-1 in both immunoprecipitates (Fig 9A, lanes 7 and 8). These bands were identified as being -actinin and TSP-1 by Western immunoblotting of the immunoprecipitates similarly obtained from unlabeled thrombin-activated platelets (data not shown). Control immunoprecipitates performed with an irrelevant mouse monoclonal antibody (Fig 9A, lane 6) or a rabbit polyclonal antibody from preimmune serum (Fig 9A, lane 10) did not contain any significant amount of radiolabeled bands except for an unidentified band of 38 kD that was present in all immunoprecipitates carried out with the polyclonal antibodies (Fig 9A, lanes 7 through 10). This band did not correspond to actin, in which electrophoretic migration is slower, as demonstrated by immunoblotting of a platelet lysate with the polyclonal antibody to actin (see Fig 9B, lane 5).

These experiments demonstrated the formation of molecular complexes between -actinin and TSP-1 on the surface of activated platelets that could be immunoprecipitated in Triton X-100–solubilized samples with polyclonal antibodies to either of these proteins.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have previously reported on a specific interaction between platelet -actinin and TSP-1 in an in vitro system.²⁷ This finding was intriguing because -actinin is believed to have a distinct platelet subcellular localization than TSP-1^{11 45 46 47 52} and because its proposed role relates to its capacity to cross-link F-actin and anchor actin bundles in the plasma membrane.^{29 44} We studied the physiological significance of the interaction between -actinin and TSP-1 in this work by looking for a possible subcellular colocalization of these proteins in platelets by using immunofluorescence and immunoelectron microscopy. We were able to demonstrate that (1) -actinin, in addition to its presence in the cytosol, is located in the -granules of resting platelets, (2) during platelet activation, the -granular fraction of -actinin is transported to the plasma membrane along the membrane of the OCS, where TSP-1, which is located in the matrix of the -granules, is also seen to concentrate before being expressed on the plasma membrane, and (3) -actinin is finally expressed on the outer surface of thrombin-activated platelets, some being colocalized with TSP-1. Furthermore, through the use of immunoprecipitation experiments of ¹²⁵I-surface–labeled, thrombin-activated platelets, the presence of molecular complexes of -actinin and TSP-1 on activated platelets was demonstrated.

Using immunofluorescence microscopy, Debus et al (1981)⁴⁵ have reported on the redistribution of cytoskeletal proteins in platelets upon cell activation by surface contact. The authors described a relatively homogeneous distribution of -actinin throughout the entire body of resting platelets, which, upon platelet activation, was found in submembraneous areas and pseudopods. However, examination of their immunofluorescence pictures suggests that -actinin is not entirely cytoplasmic, and its association with platelet secretory granules could be suspected by the granular appearance in resting platelets and its association with the centered secretory granules in fully spread platelets. Using immunoelectron microscopy to localize -actinin in resting platelets, Puszkin et al (1985)⁴⁶ described some association of -actinin with -granules, while Sixma et al (1989)⁴⁷ in their ultrastructural study on resting and thrombin-activated platelets detected a unique localization of -actinin in the cytoplasm that redistributed to the plasma membrane and pseudopods upon cell activation. These discrepancies may be attributed to the specificity of the different antibodies used in these studies. Thus Puszkin et al⁴⁶ used an antibody prepared against human platelet -actinin, whereas antibodies prepared against skeletal muscle -actinin from bovine and porcine origin were used by Debus et al⁴⁵ and Sixma et al,⁴⁷ respectively. In addition, two different isoforms of -actinin have been described in platelets that differ in their structural and immunologic properties as well as calcium sensitivity with respect to F-actin binding.^{32 33} Therefore, antibodies made against skeletal muscle -actinin may not react with both isoforms of platelet -actinin and hence the subcellular distribution of -actinin would be misleading. In this study, both antibodies used, a monoclonal and a polyclonal one, were prepared against platelet -actinin and reacted with all platelet isoforms of -actinin.^{27 32} These antibodies clearly detected -actinin in the -granules of resting platelets by electron microscopy, whereas -actinin in the cytosol was less readily detected. Since these antibodies were shown to strongly react with -actinin purified from platelet cytosol (compare with Fig 1), this observation may relate to a higher dilution of the antigen in the cytosol compartment as compared with the -granular fraction. Alternatively, the interaction of -actinin with actin in the cytosol might impair its reactivity with the antibodies. Using a biochemical approach, we estimated that 70% of -actinin was associated with the actin cytoskeleton of platelets, which attested for the existence of a large pool of cytosolic -actinin.

Upon platelet activation by thrombin, we observed that both cytosolic -actinin and actin were redistributed to the plasma membrane, but no close localization of these two proteins was noted by double immunogold labeling. This is in agreement with our biochemical studies showing that in contrast to other actin cytoskeleton-associated proteins such as myosin or filamin, there was no further incorporation of -actinin into the reorganizing actin cytoskeleton upon platelet activation under nonstirring conditions. Indeed, previous studies have demonstrated that -actinin incorporation into the actin cytoskeleton was markedly increased only when platelets were aggregated,^{43 53} that is, under stirring conditions that did not correspond to our experimental conditions. In addition, interaction of -actinin with components of the plasma membrane such as lipids,^{34 35 36 37} integrins,³⁸ or membrane-associated proteins^{36 54} may occur independently of its capacity to anchor F-actin bundles to the plasma membrane.⁵⁵

The surface exposure of -actinin on thrombin-activated platelets was demonstrated in this study by preembedding immunogold labeling and radioimmunoprecipitation studies of ¹²⁵I-surface–labeled, thrombin-activated platelets. At low concentrations, thrombin induces the extracellular release and surface expression of several components from the -granules, which occurs through the fusion of -granule membranes with the channels of the OCS connected with the platelet surface.^{56 57 58} During this exocytotic process, some -granule components may reach the platelet surface already bound to the plasma membrane rather than being released in the medium and then bound to the membrane.^{13 14 59 60} Immunocytochemical studies have pointed to the role of the OCS, originating from plasma membrane invaginations, as a privileged compartment for early interaction of -granular components with the plasma membrane.^{56 61 62} Such a membrane exposure process may well apply to the -granular -actinin; we did not detect significant amount of the protein in the extracellular medium of thrombin-activated platelets (V. Dubernard and C. Legrand, unpublished data, 1995). The bulk of -granular -actinin redistributing to the membrane of the OCS upon cell activation could be relevant to the ability of -actinin to interact with a membrane component. Interestingly, TSP-1, which is initially located in the matrix of the -granules in resting platelets, was shown to rapidly associate with the membrane of the -granules and/or the OCS upon platelet activation, then following the same route as -actinin to reach the platelet surface. A fraction of -actinin and TSP-1 molecules expressed on the platelet surface were shown to localize closely by postembedding double immunogold labeling, especially in areas of the plasma membrane corresponding to sites of OCS discharge. Furthermore, preembedding experiments showed -actinin to be preferentially localized on pseudopods, corresponding to externalization of the OCS, where TSP-1 was previously shown to concentrate.^{52 63 64} Finally, a coprecipitation of -actinin and TSP-1 was obtained in radioimmunoprecipitation experiments that demonstrated the existence of molecular complexes of these proteins on thrombin-activated platelets. In these experiments, it is unlikely that the membrane expression of -actinin was the result of a cell damage because we did not detect any trace of radiolabeled actin, the most abundant platelet cytosolic protein, on the platelet surface, and no release of lactate dehydrogenase was quantified upon thrombin stimulation. Moreover, the absence of -actinin in the supernatant of thrombin-activated platelets, as we mentioned above, is a further indication that no significant cell lysis was occurring during our experimental procedure. All together, these results strongly suggest that -actinin that becomes expressed on activated platelets originates in the -granules. We have attempted to quantify the membrane expression of -actinin on thrombin-activated platelets by using ¹²⁵I-radiolabeled antibodies. Unfortunately, the results we obtained were unreliable either because the amount of -actinin molecules expressed on activated platelets is low and/or -actinin in molecular complexes is not readily accessible to the antibodies on the cell surface. Very likely for this reason, we also failed to inhibit TSP-1 surface expression by using the anti–-actinin antibodies. However, on the basis of our ultrastructural studies, an association between -actinin and TSP-1 may actually take place in the OCS well before exposure of these molecules on the platelet surface and may not be inhibited by exogenously added antibodies. On the other hand, in functional experiments, we have observed that the monoclonal antibody used in this study has the ability to inhibit platelet aggregation (V. Dubernard. and C. Legrand, unpublished data, 1995). Because this effect was not observed with all platelet preparations, it should be interesting to try correlate this biologic response with the level of surface expression of -actinin upon platelet activation.

In conclusion, this study demonstrates the presence of the cytoskeletal protein -actinin in the -granules of human platelets and its translocation to the cell surface simultaneously with the -granular glycoprotein TSP-1 upon cell activation. Whether -actinin may be implicated in the surface expression and/or the biologic properties of TSP-1 requires further investigations. Also, the mechanism of incorporation of -actinin within the -granules should be considered. It was suggested that -granules arise from the trans-Golgi complex in the megakaryocyte, the bone marrow precursor of platelets. Whether -actinin and TSP-1 could be synthesized simultaneously and targeted as a molecular complex to the -granules has to be explored.

	Selected Abbreviations and Acronyms

 

BSA = bovine serum albumin GP = glycoprotein Ig = immunoglobulin OCS = open canalicular system PBS = phosphate-buffered saline TSP = thrombospondin

	Acknowledgments

 
This work was supported by the Institut National de la Santé et de la Recherche Médicale and the Association Claude Bernard. The authors wish to thank Dr Suzanne Menashi for her valuable criticisms and suggestions on the manuscript. They also thank Elizabeth Savariau for the photographic art.

Received January 27, 1997; accepted May 28, 1997.

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